Neuroprotective effect of sodium alginate against chromium-induced brain damage in rats

Oral exposure to chromium hexavalent [Cr(VI)] has disastrous impacts and affects many people worldwide. Cr(VI) triggers neurotoxicity via its high oxidation potential by generating high amount of ROS. Meanwhile, alginates are known by their chelating activity and ability to bind heavy metals and toxins, in addition to their antioxidant, anti-inflammatory, and anti-apoptotic activities. So, this study aimed to explore the neuroprotective potential of sodium alginate (SA) against cellular injury, DNA damage, macromolecule alterations, and apoptosis induced by oral ingestion of Cr. Forty Wistar male rats were divided into 4 groups; group I: standard control ingested with the vehicle solution, group II: Cr-intoxicated group received 10 mg/kg b.w. of potassium dichromate orally by gavage and kept without treatment, group III: SA group in which rats were orally exposed to 200 mg/kg b.w. of SA only, and group IV: SA-treated group that received 200 mg/kg b.w. of SA along with Cr for 28 consecutive days. Neurotransmitters such as Acetyl choline esterase (AchE), Monoamine oxidase A (MAOA) concentrations, Dopamine (DA) and 5-Hydroxytryptamine (5-HT) levels were assessed in brain homogenate tissues. Neurobiochemical markers; NAD+ and S100B protein were investigated in the brain tissues and serum, respectively. Levels of HSP70, caspase-3, protein profiling were evaluated. DNA damage was determined using the Comet assay. Results revealed a significant reduction in the AchE and MAOA concentrations, DA, 5-HT, and NAD+ levels, with an increase in the S100B protein levels. Cr(VI) altered protein pattern and caused DNA damage. High levels of HSP70 and caspase-3 proteins were observed. Fortunately, oral administration of SA prevented the accumulation of Cr in brain homogenates and significantly improved all investigated parameters. SA attenuated the ROS production and relieved the oxidative stress by its active constituents. SA can protect against cellular and DNA damage and limit apoptosis. SA could be a promising neuroprotective agent against Cr(VI)-inducing toxicity.


Introduction
alginates. Their polymer architectures contain carboxyl and hydroxyl groups, which make them promising for attaching heavy metals including lead, cadmium, and chromium.
Because of its water solubility, sodium alginate (SA) is one of the monovalent salts of alginic acid that is more suitable in biological systems than alginic acid [24]. Because SA is regarded as a biocompatible, non-immunogenic, and safe agent, it's non-toxic effects on vital organs are the key advantages of its use [25]. Furthermore, SA has been shown to exhibit enormous and vital biological activities, including antifungal [26], antidiabetic [27], antihypertensive [28], antioxidant [29], antiinflammatory [30], and antitumor activities [31,32]. However, its neuroprotective properties are yet to be discovered. Therefore, the present study was intended to figure out how Cr causes neurotoxicity, and to investigate the neuroprotective effects of SA in Cr-intoxicated brain tissues against cellular injury, apoptosis, and DNA damage.

Experimental animals
Forty adults male Wistar rats (10-12 weeks old) weighing 150-175 g were obtained from the breeding section of the Medical Research Center, Faculty of Medicine, Ain Shams University, Cairo, Egypt. Rats were housed for one week before starting the experiment to allow them to acclimate and had free access to food and water. The temperature was kept at 23±2˚C with a relative humidity of 55±5%. The light was set to a light-dark cycle of 12:12-hr. The experimental protocol was carried out in accordance with the guidelines issued by the Ethical Committee for animal studies of Faculty of Science, Ain Shams University, with the consent of the local institutional animal ethics committee.
Experimental design. Following the acclimation phase, the animals were randomly divided into 4 groups of 10 rats each, as follows: Group I (standard control; NC): animals received physiological saline orally for 4 consecutive weeks. Group II (Cr-intoxicated group): rats received orally 10 mg/kg b.w./day from potassium dichromate dissolved in saline for 4 consecutive weeks, as described in the previous work [33]. Group III (sodium-alginate group; SA): rats received 200 mg/kg b.w./day of SA (dissolved in saline) orally by gastric gavage for 4 weeks [34]. Group IV (SA-treated group; Cr+SA): animals were received orally 200 mg/kg b. w./day of SA solutions along with potassium dichromate.
Blood and tissue sampling. At the end of the experiment, the animals were fasted overnight and sacrificed via sodium pentobarbital inhalation. Blood samples were collected by a cardiac piercing procedure and allowed to clot for 30 min at room temperature before being centrifuged for 15 min at 3,000 rpm. For biochemical analysis, the collected sera were kept at -20˚C.
Brain tissues were removed, weighed, and rinsed in cold saline. A weight of 100mg was used for the Comet assay to determine the DNA damage, while the remaining part was minced with ice-cold PBS (0.01mol/L, pH 7.0-7.2), and tissue homogenates were then used for neurobiochemical investigations using several assays.

Determination of Cr residues in brain tissues
In order to assess the deposition of Cr in brain tissues after oral exposure to 10 mg/kg/day for 4 consecutive weeks, the levels of Cr were evaluated by atomic absorption according to the previous method [35].

Assessment of brain injury and cellular damage
Neurotransmitters and related neuronal parameters. Various neurotransmitters, as well as their metabolites and related enzymes were analyzed throughout this investigation to assess Cr-induced neurotoxicity. The concentrations of acetyl choline esterase (AchE; Cat. No. SEB447Ra) and monoamine oxidase A (MAOA; Cat. No. SEB954Ra) were measured using the sandwich ELISA assay kit purchased from Cloud-Clone Corp. Katy, USA. Dopamine (DA) and 5-hydroxytryptamine (5-HT) determinations were carried out in brain tissue homogenates by HPLC technique [36]. Briefly, tissues were homogenized in 10M ice-cold perchloric acid solution containing 0.4mM sodium metabisulphite using an ultrasonic cell disruptor (Vibracell 72434, Bio block, IIIKrich-Cedex). At 4˚C, homogenates were centrifuged for 25min at 10,000rpm. The supernatants were then filtered with 0.2μM filter (Sigma) and 1μl of the filtrate was subjected to the HPLC system that included a quaternary gradient delivery pump model HP1050 (Hewlett Packard), a sample injector model 7125 (Rheodyne-Berkeley), and an analytical column OCD 2C 18.4 X 250mm particle size 5m (Hewlett-Packard) [37]. Finally, the amount of DA and 5-HT was determined by comparison with freshly prepared standards, and their concentrations were expressed as ng/gm in brain homogenates.
Determination of NAD + levels. The Nicotinamide Adenine Dinucleotide (NAD + ) level was evaluated in brain homogenate tissues of all experimental animals by subjecting the prepared brain samples to the HPLC system in order to assess the cellular damage following Crinduced brain injury and to evaluate the therapeutic strategy of SA [37].
Neurotrophic protein marker. S100-Calcium Binding Protein B (S100B) was tested in the serum of the study groups using a commercially available sandwich ELISA kit (Cat. No. SEA567Ra) from Cloud-Clone Corp. (Katy, USA), as directed by the manufacturer. S100B protein concentrations were expressed as ng/ml of S100B in serum.

Protein profiling of brain homogenates by SDS-PAGE
According to the previously reported approach [38], soluble proteins were isolated from brain tissues collected from all study groups. Brain tissues were crushed in the frozen state for 10 min at 70˚C with continuous shaking in a 2% SDS sample buffer. At 4ºC, the resulting mixture was centrifuged for 15 min at 15,000rpm. To check that all loaded samples had the same protein concentration, the supernatant was extracted and measured for protein content using the BCA protein colorimetric assay kit (Cat. No. E-BC-K318-M, Elabscience, USA). The samples were then separated onto 12% SDS-PAGE, stained with Coomassie blue and destained for visualization.

Determination of DNA damage in brain tissues
Evaluation and quantification of the Cr-induced DNA damage in brain tissue were conducted with the Comet assay (single cell gel electrophoresis, SCGE). Briefly, 100mg of brain homogenate tissues were chopped in prechilled buffer (0.075M NaCl and 0.024M Na 2 EDTA) before being gently homogenized with a homogenizer (Ikemoto Scientific Technology Company Ltd., Japan). The samples were centrifuged for 10min at 4˚C at 15,000 rpm, then re-suspended in cold buffer. At 37˚C, 100μL of cell suspension were mixed with 200μL of 2% low melting temperature agarose and then placed on a slide that had been precoated with a thin layer of 0.5% normal melting agarose. To achieve a homogeneous layer, the cell suspension was immediately covered with a cover glass, and the slides were kept at 4˚C until the agarose solidified. To eliminate any proteins, the cells were lysed in a lysing solution (2.5M NaCl, 100mM EDTA, 10mM Tris, 1% Triton X-100, pH 10) for one hour. The slides were placed in a horizontal gel electrophoresis chamber after being washed in distilled water. Cold electrophoretic buffer (1mM EDTA, 300mM NaOH, pH 13) was added to the chamber, and the slides were maintained at 4˚C for 40 min to allow the DNA to unwind prior to electrophoretic migration. Following electrophoresis, the slides were washed three times with neutralizing buffer (0.4M Tris, pH 7.5), stained with Ethidium Bromide, then destained with distilled water for analysis [39].
A fluorescent microscope was used to examine the slides. By scanning digital images in an image analyzer (Comet V image analyzer software), the comet tail length (TL), which reflects the DNA migration length, was determined. Divide the DNA fraction in the tail by the quantity of DNA in the nucleus multiplied by 100 to get DNA tail intensity (TI). Finally, the DNA tail moment (TM) was calculated by multiplying the DNA quantity in the tail by the TL [40].

Stress-response marker and apoptotic assay
The concentration of Heat Shock Protein 70 (HSP70) in brain homogenate tissues was measured using ELISA Kit (Cat. No: E90873Ra, Uscn Life Science Inc., Wuhan, China) to assess the effects of Cr-induced stress on brain tissues and the modulatory effects of SA against this stress. In addition, the apoptotic alterations associated with Cr-induced neurotoxicity in brain tissues of all study groups were evaluated using the Rat Caspase-3 ELISA kit (Cat. No: CSB-SEA626Ra) purchased from Uscn Life Science Inc. Wuhan, China, according to the manufacturer's instructions.

Statistical analysis
The obtained data were statistically analyzed using the Statistical Package for Social Science (SPSS 1 Chicago, IL, USA) software program, version 23.0 for Windows. For quantitative parametric data, representative data was expressed as the mean ± standard error (SE) of replicate determinations. Statistical analysis was performed using analysis of variance (ANOVA) to test for variations in the means of variables between groups. p�0.05 values were considered as significant results, meanwhile p�0.01 and p�0.001 were considered as highly significant results.

Deposition of Cr in brain tissues of studied groups
Results observed in Fig 1 revealed a highly significant elevation (p�0.001) in the Cr level distributed in the brain tissues of Cr-intoxicated group compared to the standard control group. This indicates the deposition of Cr in the brain after oral exposure to the chosen dose. Importantly, when compared to Cr-intoxicated rats, these accumulated levels were significantly reduced (p�0.01) in the brain tissues of Cr + SA-treated rats. Meanwhile, no changes in the Cr levels were detected in the SA group compared to the standard control group.

Brain injury and cellular damage
Ameliorative effect of SA on neurotransmitter levels in Cr-induced brain damage. The effects of Cr and SA on some investigated neurotransmitter levels in experimental rats´brain homogenate tissue were evaluated. Results of the Cr-intoxicated group showed a significant reduction in AchE concentration (p�0.001) and 5-HT levels (p�0.005) compared to the control group. In addition, a significant decrease was detected in the DA and MAOA levels (p�0.05) in the same group. On the other hand, oral administration of SA in parallel with Cr neutralized these effects and exhibited substantial increase in all studied neurotransmitters (p�0.05) compared to the Cr-intoxicated group. Obviously, group III received only SA showed no significant change in the examined parameters compared to the standard control one (Fig  2A-2D).
Effects of Cr and SA on the selected neurobiochemical and neurotrophic markers. The selected neurobiochemical markers; NAD + and S100B protein were investigated in the brain tissues and serum, respectively, of all studied groups. Results revealed significant decrease in the NAD + level (p�0.001) detected in the brain homogenate tissues of Cr-intoxicated group compared to the normal control. However, group IV (Cr+SA) exhibited a substantial increase (p�0.001) in NAD + levels compared to the Cr-intoxicated group. Consistently, the cellular damage was demonstrated by significant increase (p�0.001) in S100B protein level in the Crintoxicated group compared to the normal control. This elevated level was significantly reduced (p�0.05) to an acceptable extent upon oral ingestion with SA in the serum of Cr+SA group. Collectively, the SA group recorded no significant changes in both the NAD + and S100B levels compared with the standard control group (Fig 3A and 3B).

Protein pattern in brain tissues by SDS-PAGE
Data represented in Fig 4A and 4B and Table 1 depict the effect of Cr and SA on the pattern of proteins in the brain tissues of the studied animal groups. Notable changes in the total number of separated bands as well as their molecular weight were detected in the Cr-intoxicated group, compared with the normal control. Additionally, the percentage of all bands was shown to be reduced to a great extent in the Cr-intoxicated group, when compared with the control group, indicating the involvement of Cr in the cellular proteins damage. Meanwhile, group IV (Cr+SA) brought somewhat all the pattern to the normal control, which implies the modulatory impact of SA administration against Cr-induced protein modifications.

Effects of SA against Cr-induced DNA damage in brain tissues
The Cr-induced DNA damage in brain tissues as well as the modulatory impact of SA against this damage were evaluated and determined using the Comet assay. Results of this assay showed significant increase in tail length, % of tail DNA with dramatic elevation in tail moment, compared to the normal control group. On the contrary, treatment with SA

PLOS ONE
significantly reduced all the parameters of Comet assay, indicating its counteracting effect on Cr-induced DNA damage. Notably, no DNA changes were detected in the group of animals received SA only (Fig 5a-5d and Table 2).

Effects of Cr and SA on stress-response and apoptosis
The expression levels of HSP70 and caspase-3 in the different studied groups reflect the stress and apoptotic-inducing effects of Cr and the role of SA in improving these observations. As  shown in Fig 6A and 6B, Cr-intoxicated group exhibited dramatic increase in both HSP70 and caspase-3 proteins level, compared with the control group. Notably, the oral administration of SA along with Cr showed a slightly non-significant reduction in the expression of HSP70 protein, while the expression of caspase-3 was significantly reduced (p�0.05) upon administration of SA in group IV, compared to the Cr-intoxicated group. Obviously, the SA group showed no significant changes, as compared to the normal control.

Discussion
This study sought to develop a novel natural-based neuroprotective agent against Cr-induced brain damage following oral exposure to Cr(VI) in the form of potassium dichromate. SA was chosen for this purpose because of its numerous bioactivities, which allowed us to investigate its effects on achieving this goal. Following oral ingestion, Cr(VI) is normally subjected to reduction in the gut in order to be detoxified. However, a small percentage can escape or bypass the gut's modulatory function, and unfortunately, be deposited in vital organs such as liver, kidney, lung, and brain [14]. The present study focuses on evaluating the toxic impacts of accumulated Cr in the brain tissues of rats following oral ingestion a dose of 10 mg/kg b.w. for 28 consecutive days. Higher Cr residues were detected in the brain homogenate tissues of the Cr-intoxicated group compared to the unexposed animals, indicating the ability of Cr to accumulate in the brain tissues and trigger neurotoxicity [41]. On the other hand, SA was shown to alter the biodistribution of Cr  (VI) and reduce its levels in brain tissues, as evidenced by the notable reduction in the Cr level in the brain tissues of Group IV (Cr+SA) compared to the Cr-intoxicated group. It has been previously proposed that SA has the chelating and biosorption capacity to bind to Cr (VI) in the intestine, and thus prevents its accumulation in brain tissues [42].
It has been reported that oral exposure to Cr(VI) induces many neurochemical changes. Unfortunately, Cr(VI) can cross the blood brain barrier, causing oxidative stress and triggers alterations in the metabolism of several proteins implicated in the neurodegeneration process [20]. AchE catalyzes the breakdown of acetylcholine, which function as a neurotransmitter, and is linked to a variety of cognitive processes via cholinergic receptors activation [14]. Cr (VI) induces neurotoxicity by inhibiting the AchE activity through binding to its anionic active site, thus preventing the acetylcholine from binding to the enzyme, or by affecting the process of AchE synthesis [43]. This leads to the excessive accumulation of acetylcholine neurotransmitter, which in turn disrupts the nerve functions, and even may lead to death [44]. The current finding revealed a significant decrease in the AchE concentration in the brain tissues of Cr-intoxicated group, confirming that Cr(VI) can be considered as a neurotoxic contaminant by affecting the cholinergic signaling. In line with our results, Ciacci et al.
[45] reported a considerable reduction in AchE of Mytilus galloprovincialis exposed to Cr(VI). In addition, it has been postulated that oral exposure to Cr(VI) at a dose over 60 mg/kg significantly inhibited the AchE activity in brain tissues of S. schlegelii [44].
Serotonin, commonly known as 5-hydroxytryptamine (5-HT) is the main monoamine neurotransmitter involved in numerous process. Hence, disturbance in 5-HT levels is responsible for many neurological diseases and neuropsychiatric disorders, so it was selected in the current study as a potent neurobiochemical marker. Previous studies proved the high affinity of Cr to bind to the 5-HT 1A receptors, which regulates 5-HT release in neurons via the negative feedback mechanism, affecting postsynaptic events and 5-HT release [46]. These results are in agreement with our findings and demonstrate that Cr(VI) can be considered as a serotoninergic neurotoxin, as indicated by the significant decrease in the 5-HT levels detected in the Crintoxicated group compared to the standard control group.
Dopaminergic neuronal cells were also affected by oral exposure to Cr(VI). DA is considered as one of the most studied neurotransmitters in the brain [47]. Therefore, we examined the level of DA since it is implicated in many neurodegenerative diseases. DA catabolism, both enzymatic and non-enzymatic, is hypothesized to be capable of generating harmful ROS. DA oxidation can occur either spontaneously in the presence of metal ions or through enzyme-catalyzed monoamine oxidase (MAO) reactions [48]. Measurement of DA levels in the present study showed obvious reduction in the Cr-intoxicated group, compared to the standard control one. These results are in agreement with the previous studies, that recorded a significant decrease in DA along with a significant inhibition in AchE activity in the brain of Drosophila larvae that were fed Cr(VI)-salt-mixed diet accompanied by deficit in motor activity with poor balance, proving the redox-mediated influence of Cr (VI) on dopaminergic neurons [49]. Earlier study reported the Cr-induced cell death in SH-SY5Y neuroblastoma cells (a dopaminergic neuronal-like cell model) that used to study neurotoxicity [50].
Interestingly, the reduced levels of DA in the current study are concomitant with the diminution of MAOA concentration upon oral exposure to Cr(VI). It has been previously shown that MAOA levels play a vital role in the regulation of biogenic amines levels and neural activity. Another study [51] reported a decrease in the MAOA concentration in the cortex, striatum, and cerebellum following exposure to heavy metals, which is consistent with our findings. This suppression could be attributed to the heavy metals' capacity to bind to the cysteine residue of the MAOA enzyme, leading to the disruption of its structure, and hence, decreasing its levels [52]. Furthermore, the alterations in MAOA concentration have significant role in initiating cell apoptosis through ROS generation [53]. Collectively, Cr(VI) can trigger neurotoxicity via reducing the concentrations of AchE and MAOA, as well as the levels of 5-HT and DA. By preventing the neurotoxic effect of Cr through regulating the levels of neurotransmitters, SA administration along with Cr(VI) restores to a great extent the AchE and MAOA concentrations and returns the detected reduction in the 5-HT and DA levels to almost their normal ranges. This could be because SA is enriched with fucoidans, a sulfated polysaccharide with ROS and RNS scavenging and chelating properties, as well as the ability to suppress lipid peroxidation. This is endorsed the SA as a potential therapy for neurodegenerative diseases associated with ROS and RNS overproduction [54]. Moreover, through enhancing AchE level, fucoidan has been shown to have a potential therapeutic role in improving the cognitive impairment [55]. Following brain injury and during the progression of neurodegenerative diseases, mitochondria generate free radicals that can trigger further cellular damage. Meanwhile, the decline in NAD + levels following brain injury promotes the production of ROS and limits the ability of mitochondria to repair damage, which leads to further accumulation of these free radicals in cells. In the present study, the significant decline detected in the NAD + levels in the brain tissues of Cr-intoxicated group confirmed the occurrence of brain injury in this group. This infers that Cr(VI) induced toxicity via inhibiting the mitochondrial dehydrogenases activity such as NADH dehydrogenase, which in turn causes depletion of NADH pool from the tissues [56]. Consistently, previous study postulated the neurotoxicity in the chicken brains following exposure to Cr(VI) [19]. Although there are not enough evidences available to evaluate the effect of SA on NAD + , but, the current treatment with SA restored its reduction, which may be ascribed to the appreciable antioxidant ability of SA. Accordingly, Hentati et al. [55] reported the NO scavengers' ability and antioxidant activity of SA isolated from brown alga Cystoseira compressa. Indeed, NAD + plays an important role in maintaining cellular health and repairing DNA damage. It is a vital cofactor for crucial enzymes involved in brain bioenergetics for metabolism and ATP production. Additionally, NAD + is a key substrate for various classes of NAD + -dependent enzymes implicated in neuronal integrity. This confirms the tight association between the reduction in NAD + levels and genomic stability, mitochondrial homeostasis, adaptive stress responses, and cell survival [57]. S100B protein was chosen in the current study as one of the neurotrophic proteins and as one of the potential neurobiochemical markers useful in detecting the Cr-induced brain cell injury and in exploring the efficacy of SA in improving this damage. In the Cr-intoxicated rats, the level of S100B protein increased significantly, according to the current data. It has been shown that Cr(VI) induces neurotoxicity by direct impact on the S100B protein expression, since high S100B protein levels increase the expression of β-amyloid precursor protein and its mRNA in brain tissues. In turn, these β-amyloid plaques enhance the synthesis of more S100B proteins at both transcriptional and translational levels [58]. Indeed, the cellular damage could be attributed to the binding of extracellular S100B proteins with receptor for advanced glycation end products (RAGE), which activates the overproduction of ROS and the release of cytochrome-c from mitochondria and hence induces apoptosis [59]. Interestingly, the current findings ascertain the potent ability of SA to reverse the cellular damage by means of its antioxidant activities.
Being a strong oxidizing agent, Cr(VI) has the ability to interact with cellular macromolecules such as DNA and proteins. This interaction is considered one of the main leading causes of DNA damage and protein modifications [60]. The Cr-inducing DNA damage in brain tissues was further confirmed throughout the present study, as evidenced by the significant increase in comet tail length detected in the Cr (VI)-intoxicated group, compared to the standard control one. Comet tail length is an important parameter in evaluating the DNA damage in the Comet assay [61]. Cr(VI) induces genotoxicity by its intracellular reduction into Cr(III) with the generation of Cr metabolite radicals that in turn generate hydroxyl radicals, and hence, induce oxidative stress. This may lead to different forms of DNA damage; including DNA-Cr-protein crosslinks, DNA inter/intrastrand crosslinks, single-and double-strand breaks. Ultimately, this leads to many other cellular injuries, apoptosis and/or necrosis [62,63]. In line with our findings, other studies [61,64] reported the involvement of Cr(VI) in generating ROS, leading to oxidative stress following I.P. injection with potassium dichromate. Thus, removal of ROS or inhibition of their generation by using natural antioxidants to prevent the oxidative cell death has been attracting attention as an essential target for neuroprotection. Recently, researchers have made considerable efforts to search for natural antioxidants with neuroprotective potential. Previous data confirmed that treating cells with H 2 O 2 resulted in cell death which was greatly decreased in the presence of alginate. This result designates that alginate significantly protected the NT2 neurons from H 2 O 2 -induced cytotoxicity [65]. Alginate's inhibitory effect on H 2 O 2 -induced β-amyloid formation in Parkinson's disease has Previously been linked to its ROS-scavenging abilities [66].
Fortunately, the present results proved that SA has no toxicity at both the cellular and genetic levels. The existence of SA's various antioxidant scavengers, such as fucoidans, may explain its protective activity against Cr-induced genotoxicity [67]. The antigenotoxicity of fucoidan was previously identified against doxorubicin-induced DNA damage and proved in terms of chromosome aberrations and comet in human lymphocytes [68]. Accordingly, other study reported that fucoidan protects mice from 4-NQO-induced genetic damage in terms of LPO, 8-OHdG, micronuclei, comet and apoptosis [69].
Regarding the influence of Cr(VI) on the protein pattern of brain tissue homogenates, the present findings confirm the ability of Cr(VI) metabolites to intercalate with protein and lead to alterations in their size and concentrations. It is noteworthy to note that mitigating oxidative damage is considered as a promising strategy for preventing DNA and protein injury. However, SA was able to alleviate both DNA damage and protein alterations induced by oral exposure to potassium dichromate. These modulatory effects confirm the neuroprotective impact of SA against Cr(VI)-induced neurotoxicity, which may be ascribed to the ability of SA to attenuate the production of oxidative species. Previous findings stated the protective effects of alginate on D-gal-induced kidney aging by promoting the translocation of Nrf2 protein from the cytoplasm to the nucleus, where Nrf2 is associated with D-gal-induced kidney aging. Moreover, alginate increased the gene expression of Klotho protein in D-gal-induced kidneys, which reflected the protective impact of alginate-against kidney aging. In parallel, the alginate ameliorated the oxidative stress in D-gal-induced aging mice through enhancing the activities of SOD and CAT and decreasing the level of MDA [70].
In the same line, the present study revealed the Cr-induced cellular stress that was confirmed by the high level of HSP70 protein detected in the Cr-intoxicated group. Under normal physiological conditions, HSP70 is produced constitutively and play a role in protein metabolism. However, when exposed to harmful conditions such as oxidative stress, heavy metals, and mechanical stress [71], the level of HSP70 increases to protect cells from harm [72]. These findings point to a mechanism for the pathogenesis of cellular degeneration caused by high levels of HSP70 in the cell. Indeed, HSP70 expression can be a used as a protective mechanism for stressed and apoptotic neurons because it reduces protein aggregates and intracellular inclusions. According to previous results, the increased expression level of HSP70 in histological analysis after exposing chondrocytes in alginate beads to hydrostatic pressure was strong evidence for evoking apoptosis in the extracellular matrix [73]. Another study used the alginate microparticles as oral capsules for delivering recombinant Escherichia coli HSP70 as safe vehicle due to their biodegradability, low toxicity and low immunogenicity [74].
Finally, the neural cell death induced by oral exposure to potassium dichromate throughout this study points out the neurotoxicity and brain damage triggered by Cr(VI). The apoptoticinducing ability of Cr was elucidated by the significant increase in caspase-3 levels detected in the Cr-intoxicated group of rats. In accordance with these results, Catelas et al. [75] demonstrated that Cr ions can induce apoptosis via the caspase-3 pathway. On the other hand, alginate is well documented as a non-immunogenic, non-toxic, biodegradable polymer with amazing biological properties such as antioxidant, antiinflammatory, and antiapoptotic effects. Accordingly, the ability of SA to reduce HSP70 and caspase-3 levels in the current study illuminates its effective response against Cr-induced oxidative stress and apoptosis, indicating its antioxidant and thus antiapoptotic activities via decreasing the production of ROS levels. In agreement with our results, further study recommended the use of alginate oligosaccharide as a food supplement for humans or animals, as evidenced by the effect of alginate ingestion on decreasing the pro-apoptotic factors; BAX, caspase-3 and caspase-9 mRNA with concomitant increasing in the anti-apoptotic factor BCL2 mRNA in the jejunum. These findings confirmed alginate's ability to inhibit intestinal epithelial cell death, which may be due to a reduction in mitochondria-dependent apoptosis [76]. As previously reported, alginate oligosaccharide pretreatment significantly neutralized the level of cleaved caspase-12, and thus the level of cleaved caspase-3, which was nearly equal to the control in the H 2 O 2 -induced group [77].

Conclusion
This study demonstrated that oral exposure to Cr (VI) has a significant effect on rat brain tissues and induces neurotoxicity by lowering AchE and MAOA concentrations, as well as 5-HT and DA levels. The observed decrease in the NAD + levels and upregulation of the neurotrophic marker S100B proteins confirmed brain injury and cellular damage. Moreover, Cr(VI) triggers toxicity by interacting with cellular macromolecules, leading to protein modifications and DNA damage. Finally, the stress exerted by Cr(VI) and neural death were proved throughout this study by high levels of HSP70 and caspase-3 proteins. Fortunately, oral administration of SA in conjunction with Cr(VI) has been shown to prevent the accumulation of Cr in brain tissues, thereby reducing ROS production and alleviating oxidative stress. By this means, SA can significantly improve neurotransmitters and neurobiochemical parameters, protect against cellular and DNA damage, and limit apoptosis. These findings suggested that SA could be a promising neuroprotective agent against Cr-induced toxicity and brain damage.
Our obtained data show that SA is a valuable neuroprotective agent, and we recommend testing its beneficial effects in models of neurodegenerative diseases like Alzheimer's and Parkinson's. Indeed, the mechanisms underlying the neuroprotective impacts of alginate remain to be further investigated.